Duchenne muscular dystrophy (DMD) is an inherited progressive myopathy with the highest incidence which occurs at a frequency of about one in 3,500 live male births. In their infancy, DMD patients show almost the same motor function as in normal humans, but they show signs of muscle weakness around the ages of 4 to 5 years. Then, their muscle weakness progresses to the loss of ambulation until the age of about 12 years and eventually leads to death in their twenties due to heart failure or respiratory failure. DMD is such a sever disease. Currently, there is no effective therapy for DMD, and hence the development of a new therapeutic agent is strongly demanded.
DMD is known to be caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and is a huge gene consisting of 2.2 million DNA nucleotide pairs. This DNA is transcribed into precursor mRNA and further spliced to remove introns, thereby resulting in mRNA consisting of 79 exons joined together, which is 13,993 bases in length. This mRNA is translated into 3,685 amino acids to produce a dystrophin protein. The dystrophin protein is involved in maintenance of the membrane stability of muscle cells and is required to make muscle cells less prone to breakage. DMD patients have mutations in their dystrophin gene and therefore show almost no expression of a functional dystrophin protein in their muscle cells. For this reason, in the body of DMD patients, muscle cells can no longer retain their structure and an abundance of calcium ions flows into the muscle cells. As a result, a reaction similar to inflammation will occur to promote fibrosis, so that muscle cells are difficult to regenerate.
Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene. As its symptom, muscle weakness is observed, but is usually milder and progresses slower than in DMD, so that BMD develops in adulthood in most cases. Differences in clinical symptoms between DMD and BMD appear to arise from whether mutations disrupt or maintain the amino acid reading frame during translation from dystrophin mRNA into a dystrophin protein (Non-patent Document 1). Namely, DMD patients show almost no expression of a functional dystrophin protein because of having mutations responsible for shifting the amino acid reading frame, whereas in BMD patients, mutations cause deletion of some exons but the amino acid reading frame is maintained, so that a functional albeit incomplete dystrophin protein is produced.
As a therapy for DMD, the exon skipping therapy is promising. This therapy involves modification of splicing to restore the amino acid reading frame in dystrophin mRNA, thereby inducing the expression of a dystrophin protein with partially recovered function (Non-patent Document 2). Amino acid sequence regions targeted by exon skipping are deleted in this therapy. For this reason, a dystrophin protein expressed in this therapy is shorter than the normal protein, but partially retains the function of stabilizing muscle cells because the amino acid reading frame is maintained. It is therefore expected that exon skipping allows DMD to present the same symptoms as seen in BMD which is milder. The exon skipping therapy is now under clinical trial in human DMD patients after animal experiments in mice and dogs.
Exon skipping can be induced by binding of antisense nucleic acids directed against either or both of the 5′ and 3′ splice sites or against exon internal sequences. An exon is included into mRNA only when its both splice sites are recognized by a spliceosome complex. Thus, exon skipping can be induced when the splice sites are targeted by antisense nucleic acids. Moreover, to induce exon recognition by the splicing machinery, SR proteins rich in serine and arginine would be required to bind to exon splicing enhancers (ESEs); and hence exon skipping can also be induced upon targeting to ESEs.
DMD patients have different mutations in their dystrophin gene, and hence various antisense nucleic acids are required depending on the position and type of gene mutation. There are some reports of an antisense nucleic acid designed to induce exon skipping of a single exon in the dystrophin gene by targeting a single continuous sequence (Patent Documents 1 to 6, as well as Non-patent Documents 1 and 2). In addition, there is a report showing that when two different antisense nucleic acids directed against the same exon in the dystrophin gene are allowed to act in admixture (double targeting), skipping activity may be enhanced as compared to when each antisense nucleic acid is used alone (Patent Document 7).
However, there has been no report showing that connected single-stranded antisense nucleic acids directed against two or more sites in the same exon (i.e., antisense nucleic acid of connected type) show skipping activity.